Ali Brivanlou - US grants

This is a Shannon Award providing partial support for the research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon Award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. The abstract below is taken from the original document submitted by the principal investigator. The ultimate objective of this proposal is to characterize the cellular and molecular basis of vertebrate embryonic neural development. Both classical embryology and modern molecular biology will be used to isolate and characterize genes that play key roles in the formation of the vertebrate neuroaxis. The animal chosen for these studies is the frog Xenopus laevis whose large sized embryo facilitate microsurgical manipulations. The proposed studies will focus on follistatin, a specific inhibitor of activin and one of the two soluble proteins known to direct neuralization in vertebrate embryonic cells. Activin itself belongs to the TGF-beta superfamily of growth factors and has been shown to be involved in mesoderm induction in vivo. By blocking activin, through direct protein-protein interaction, follistatin changes the fate of ectodermal cells from epidermal to a neural fate. Five objectives are described for the next five years. First, we plan to isolate genes regulated by follistatin in vivo. By identifying such genes, we can map out the molecular cascade initiated by follistatin and, thus, the molecular pathway to neural induction. Second, we will address the specificity of follistatin which is generally considered an active- specific inhibitor, however has not been rigorously tested against the newly discovered TGF-beta ligands. We propose to test the effect of follistatin on other TGF-betas in the context of embryonic explants. Third, to determine the function of endogenous follistatin in the embryo, we have proposed strategies to knock-out follistatin function in vivo. Fourth, we will analyze the embryonic mechanisms of neural induction by addressing how a neuralizing signal such as follistatin diffuses and what kind of cell-cell interactions are required for the spread of the signal in vivo. Finally, a simple in vitro bioassay will be used to assess the function of peptide growth factors in patterning neural tissue. In this assay, ectoderm that has been neuralized by follistatin will be exposed to different concentrations of peptide growth factors. The neural patterning function of each factor or combination of factors will be assessed using region specific neural markers in the treated ectoderm. The isolation of factors involved in neurogenesis has immediate health related consequences. Factors which induce neural differentiation in ectodermal and mesodermal cells may provide insights into regeneration of neural tissue in adults and in the long term may provide therapy for diseases involving loss of neuronal cells, including stroke and neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS). The combination of approaches suggested in this proposal should provide opportunities to answer long-standing questions regarding the process of neurogenesis in vertebrates at the molecular, cellular and embryological level.

Although lens induction in vertebrate embryos was discovered almost a hundred years ago by Spemann and colleagues, the molecular mechanism underlying this process is still unknown. It has recently been shown that the the transcription factors six3 and Pax6 have lens inducing activity in vivo. Expression of Six3 in medaka fish can induce lenses in otic vesicle while our own work has shown that Pax6 can induce lenses in multiple locations in the frog Xenopus leavis. In a collaborative effort between the Hemmati-Brivanlou and Lang labs, we propose to unravel the molecular pathways leading to lens induction in vertebrates. We will take advantage of the powerful expression cloning approaches available in Xenopus laevis to identify candidate lens inducing factors. These candidates will then be tested for their activity in mammals using the genetic systems available in the mouse. In our preliminary results, we describe two important advances. (1) Xenopus expression cloning of a factor with lens inducing activity. This factor (designated D4E) is unique, in that no orthologue exists in the sequences databases. However, the domain structure of the full-length molecule (designated 4E) clearly indicates that it has a role in signal transduction. The lens-inducing cDNA D4E encodes an N-terminaly truncated protein that is likely to act as a dominant negative mutant of full length 4E. (2) Identification of a lens-specific enhancer derived from the Pax-6 gene. This region of DNA directs transgene expression to the presumptive lens ectoderm only starting at embryonic day 8.75. Armed with these reagents, we propose in five specific aims to further characterize the activity of our novel gene 4E. In Xenopus, we will determining in which signaling pathway it functions and inquire about a possible regulatory relationship between 4E and Pax6. In the mouse we will use the lens-specific enhancer element to target 4E expression to the presumptive lens to ask about the function of 4E in-vivo. Finally, we propose to eliminate 4E from the mouse genome to determine if 4E is necessary for lens induction in mammals in vivo. We strongly believe that the comparative approaches proposed in this grant will allow the molecular resolution of one of the oldest embryological problems. Elucidating the molecular basis of lens induction in vertebrates will inevitably have a direct consequence on the rational design of drugs for the treatment of human lens diseases.

DESCRIPTION (provided by applicant): This proposal addresses the role of TGFbeta signaling in the formation of the vertebrate mesodermal germ layer. Using Xenopus laevis embryos and embryonic explants in the context of expression cloning, candidate gene approaches and microarray technology, we have identified five genes which, by modulating the TGFbeta pathway provide a strong input in mesoderm formation. The candidate gene approach has lead to the discovery that LTBP-1 is an organizer specific gene which binds and synergizes with all the embryonic functions of activin, including mesoderm and secondary axis induction. I propose to address the specificity of the interaction of LTBP-1 with members of the TGFbeta family, its relationship with organizer specific inhibitors which also bind to activin such as follistatin, and explore the functions of related family members LTBP2 and 3. A gain of function screen has led to the discovery of OS4, an evolutionarily conserved gene of previously unknown function, which is also able to induce mesoderm and secondary axis. I propose to link OS4 to a signaling pathway(s), first by performing an epistatic analysis of OS4 in the TGFB pathway, and second by isolation of binding partners. Our prototype Xenopus microarrays have identified a large number of activin-regulated cDNAs, including three novel genes. I propose, first, to follow on the embryological and biochemical activities of these novel responsive genes. Second, I propose the use of a 5000 gene array available in my laboratory to globally identify genes (including immediate early response) that are regulated by different thresholds of activin protein concentration, with the aim of characterizing and organizing temporally the repertoire of genes regulated by activin. Taken together, the aims of this grant will enrich our knowledge not only about early vertebrate mesoderm formation, but also about the TGFBeta pathway, which is relevant to many other areas of biology and medicine beyond embryology.

[unreadable] DESCRIPTION (provided by applicant): Embryonic stem cells are pluripotent cells derived from the epiblast of the early embryo. In culture they exhibit two important properties: they can maintain an undifferentiated state indefinitely, and they can be pushed to differentiate into a variety of cell types. This proposal targets the molecular dissection of pluripotency and self-renewal in both mouse and human embryonic stem cells (MESCs and HESCs), comparatively. We have recently obtained, and successfully cultured, five of the NIH-approved HESC lines (NIH codes WA01, BG01, BG02, ES03, and ES04). Our preliminary results provide a signature for the state of stemness in both ESCs. We have also demonstrated that the activation of the Wnt pathway is sufficient to maintain the undifferentiated phenotypes in both species. I propose two specific aims: 1- Refinement of the global and comparative molecular profiling of HESCs, and its comparison with MESCs. This allows the establishment of: (i) quantitative molecular standards, for undifferentiated HESCs, (ii) characterization of causal genes for the state of stemness, and (iii) measurement of molecular changes during differentiation paradigms. 2- Characterization of the role of signaling pathways underlying pluripotency, by (i) testing the activity of a novel small compound GSK3[unreadable] inhibitor, BIO, in vivo in mouse embryos; (ii) addressing the role of TGF[unreadable] signaling in this process; and (iii) performing an unbiased expression cloning to identify molecular factors involved in self-renewal in HESCs. In addition to providing a window into early human embryogenesis, HESCs could constitute a renewable source of a large variety of differentiated cells and be employed to replace diseased or damaged tissue by cellular transplantation. Therefore, the establishment of molecular standards and global molecular knowledge of their pluripotency is an obligatory step toward the design of rational clinical treatments. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The overall goal of this proposal is to elucidate the biochemical and embryological function of the Transforming Growth Factor-B (TGF-B) pathway in the induction and patterning of the mesodermal embryonic germ layer in Xenopus. By combining expression cloning in Xenopus embryos, global transcriptional profiling, and biochemical approaches during the last round of funding, we have identified key factors which modulate the TGF-B pathway at multiple levels in the generation of the primary embryonic axis and the establishment of discrete cell fates. Building on this progress, we will focus on 2 of these factors that are unusual regulators of the TGFB pathway: Coco, acting outside of the cell, and SCP2, the first identified Smad phosphatase, acting in the nucleus. I have 2 main objectives. The first is to dissect the molecular mechanism underlying the biochemical function of coco in the context of the very early embryo. This will be done by eliminating Coco from the fertilized eggs and assess the consequence of this loss of function on the development of the embryo. In line with this mechanistic analysis, embryonic factors that interact with Coco protein will be identified to unveil Coco-partners. The second aim, targets the unraveling of the biochemical, cellular and embryological function of long awaited players in the TGFB pathway, Smad phosphatases, which we have finally identified. We will also extend this analysis to individual family members of this group by addressing comparatively their biochemical and embryological functions. The TGFB pathway has been evolutionarily conserved from C. elegans to human, covering an amazing range of biological activities both in embryogenesis and adult life. Mutations in this pathway are the causes of various diseases including developmental disorders and human cancer. Therefore, the findings derived from the studies presented in this application extend beyond their relevance to our basic molecular understanding of embryological events, and reach our knowledge about this important signaling pathway, reiterated again and again in different tissues and cell type throughout life. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The Smad2/3 branch of the TGF2 signaling pathway is necessary to sustain pluripotency in human embryonic stem cells (hESCs) and human induced pluripotent cells (hiPSCs). Despite its significance, the molecular circuitry underlying pluripotency, as well as differentiation, remains poorly understood in both systems. Recent work, including our own, has shown that embryonic specific microRNAs (miRNAs) play crucial roles in modulating the fate decisions in early vertebrate development and HESCs by modulating the Smad2/3 pathway. This pathway acts as morphogen, transducing activin/nodal signals and eliciting different outcomes based on concentration and duration of signaling. While tremendous progress has been made in the resolution of the molecular mechanism underlying miRNA biochemistry and mechanism of action, their role in biological processes, specifically during early embryonic development and cell fate determination, also remains poorly understood. The two specific aims of this grant are to bridge these two areas in order to continue our efforts toward a more comprehensive understanding of both. Preliminary experiments presented here demonstrate that different levels of inhibition of ongoing Smad2/3 signaling (mimicking morphogen effects) specifically modulate expression of several miRNAs in hESCs. Based on these findings I propose to investigate the regulatory mechanisms and the function of these Smad2/3-regulated miRNAs during pluripotency and differentiation of hESCs and hiPSCs. Our first goal is to determine the regulatory threshold of Smad2/3 signaling required for the proper expression of each miRNA, followed by the discrimination between direct versus indirect, and transcriptional versus post-transcriptional mechanisms of regulation. Target analysis for each threshold-specific miRNA will address their molecular function. Secondly, we will perform gain and loss of miRNA function directly in hESCs and hiPSCs. Taken together, the successful accomplishment of the aims proposed in this study will contribute to a more in depth understanding of the role of threshold-specific Smad2/3 activity during hESC fate acquisition when observed from the miRNA perspective, and establish the biological function of these selectively expressed miRNA in the same context. Moreover, given the similarities between cancer cells and embryonic stem cells and the established role of both the TGF2 pathway and miRNAs in tumorigenesis, the discovery and the functional characterization of relevant Smad2/3-regulated miRNAs in hESCs and hiPSCs will have a direct impact in the cancer field. PUBLIC HEALTH RELEVANCE: In this study we propose to investigate the regulatory mechanisms and the function of these Smad2/3-regulated miRNAs during both pluripotency and differentiation of hESCs. By targeting and resolving the role of the Smad 2/3 branch of the TGF2 pathway and their regulation of miRNAs in the context of hESC differentiation, these studies contribute to our basic understanding of molecular processes involved in fate determination in hESCs. Moreover, as this pathway is also involved in cancer, the resolution of the aims of this study will also contribute to our basic understanding of the molecular basis of tumor formation. Thus, the results obtained from the successful accomplishment of the aims of this grant will have a significant impact on biomedical science and public health.

DESCRIPTION (provided by applicant): The transitions from egg to embryo to adult require that cells communicate with each other to acquire specialized fates at the right times in the correct places. Cancer is in part the result of aberrant regulation. Most of our information on signaling pathways comes from genetics and biochemistry on populations of cells, with stimuli typically reduced to a binary on/off. This project will adapt contemporary microfluidic technology to accurately control the concentrations of signaling molecules in time in multiple small chambers while continuously imaging the response of single cells via fluorescently tagged proteins that move in response to signals. We will use human embryonic stem cells (hESC) for this purpose because of their relevance to regenerative medicine. We will stimulate the cells with ligands from the TGF¿ pathway since they can induce the first round of developmental choices these cells naturally make. This pathway has two sub-branches that interfere, making it a natural context in which to study pathway interactions. Our data will be organized and developed into a predictive tool with which to rationally reprogram specialized fates from hESCs.

DESCRIPTION (provided by applicant): Our knowledge on the molecular basis of pluripotency and differentiation remains limited. This is mostly due to the fact that molecular and genetic tools developed in model systems are mostly lacking in human embryonic stem cells (hESCs). To overcome this shortcoming, I propose to use a transposon mediated genetic screen directly in hESCs to identify mutants and isolate genes that control the ability of human cells to differentiate. To perform our screen we will use a genetically marked hESC line, RUES2 (originally derived and characterized in my laboratory and part of the NIH registry), that has a single copy of two types of insertions: (i) a stable lentiviral insertion where the human Oct4 promoter drives the expression of EGFP and Neomycin (Neo), (ii) a mutagenic transposable element equipped with a transcriptional amplifier cassette and Hygromycin (Hyg). We have previously shown this transposable element can be mobilized to hop and randomly insert into the human genome. We propose to take advantage of this property to generate mutations and perform an unbiased forward genetic screen, as described in model organisms. Using a very simple screening strategy, based on positive (EGFP) and negative selection (Neo and Hyg), 107 hESCs will be mutagenized and challenged for their ability to retain pluripotency and lose their ability to differentiate. The transposon has properties that allow the characterization of th mutated gene with a straightforward plasmid rescue strategy. Additionally, it can be excised from the human genome without leaving a footprint to generate revertants. Those, in addition to phenocopies of the mutant by independent means, will confirm the link between the gene and the mutant phenotype. This large-scale genetic screen will lead to the isolation of genes that control a cell fate decision in hESCs. As hESCs provide a window to early human embryogenesis, this knowledge will move us a step closer to the understanding of our own development. More importantly, the successful accomplishment of this project will open the door to a more systematic use of unbiased genetic screens in human cells. In turn, this will have a strong impact on both basic research and clinical applications.

DESCRIPTION (provided by applicant): The allocation of cells into the three early embryonic germ layers, in vivo, follows a specific patio-temporal sequence. In response to growth factors, human embryonic stem cells (hESCs) can generate the three germ layers in culture, but differentiation is random and spatially disordered. We have demonstrated that when hESCs are grown on micro-patterned surfaces in colonies of defined size and shape, they differentiate with quantitatively reproducible spatial patterns of gene expression suggestive of those in mammalian embryos. This is the first demonstration of ordered germ layer patterning in vitro and opens the door to investigations of early development currently impossible to perform in intact mammalian embryos, particularly human embryos. Since the spatial pattern develops spontaneously in a well-mixed medium, this system affords a quantitative phenotype with which to analyze cell-cell signaling. The goal of this grant is to establish micro patterned cell cultureas a system for studying signaling dynamics and spatial patterning in hESCs. We propose to further validate our assay by differentiating mouse epiblast stem cells (the closest analogue to hESC) on our substrates and comparing with mouse embryos purchased from commercial sources. We will stimulate the TGF-ß, BMP, and Wnt pathways that pattern the early mammalian embryo and follow the transcriptional effectors for these pathways as well as the resulting pattern of germ layer markers in time. We will engineer cell lines with combinations of fluorescent reporters for signaling and fate and use these for time-lapse imaging. The results will be fit to a phenotypic computational model that will permit us to rationally design combinations of ligands with defined doses and timings that produce a defined outcome. We will also manipulate the levels of secreted ligands (both activators and inhibitors) that are responsible for paracrine signaling in the embryo on our micro patterned colonies and further quantify cell-cell interactions. Physical assays for secreted ligands using microfluidics will be employed to quantify mechanisms of ligand transport.

Project Summary During early embryogenesis, pluripotent cells specify their fates by integrating multiple signals delivered at different times, places, and with different dynamics. In the mouse embryo, interplay between 3 signaling pathways - BMP4, Wnt, and Nodal - initiate the induction and patterning of embryonic germ layers. However, the relevance of these pathways to human development is not understood. Furthermore, how multiple dynamic signaling pathways are integrated to define cellular fate is unclear. Here we propose to use human embryonic stem cells (hESCs) to understand how individual cells process these dynamic signals to generate discrete fates. To address this, we developed 2 innovative technologies. First, microfluidics to precisely control the timing of ligand application and follow the behavior of the signal transducer SMAD4, with which we demonstrated that TGF? signaling was adaptive. Second, micropatterns to control hESC colony size and geometry, to show that in response to BMP4, hESCs cultured in circular colonies self-organize into radially symmetric patterns of discrete embryonic germ layers. This remarkably recapitulates the proximal-distal axis of the gastrulating mouse embryo. In this competitive renewal, we combine the strengths of both technologies to deliver distinct dynamics of ligand presentation by microfluidics to CRISPR-edited hESC lines cultured in micropatterned colonies. Four independent signaling-reporter hESC lines that fluorescently tag SMAD1, SMAD2, SMAD4, and ?-CATENIN will be used to visualize signaling, and one triple-tagged, fate-reporter CRISPR-edited line that fluorescently tags SOX2, BRACHYURY, and SOX17, will be used to monitor fate acquisition. Our CRISPR-reporter lines will be used to measure signaling dynamics and fate acquisition, with single cell resolution and in real-time by video-microscopy, when cells are presented with BMP4, Wnt3A, and Activin/Nodal either as a persistent step of defined concentration, or as one of defined duration. We propose three specific aims. In aim1, the three ligands will be presented to our signaling-reporter CRISPR lines, to follow the behavior of the four tagged signal transducers and to determine the dynamic behavior of each pathway. In aim2, using the same approach, we will evaluate pathway output by measuring the activity of transcriptional reporters for the three ligands, and use our triple fate-reporter CRISPR line to follow fate acquisition. These two approaches will establish a quantitative link between signaling dynamics, transcriptional output, and fate determination. In aim3, large datasets obtained from aims1 and 2, will be used to model the kinetics of signal transduction, and provide a mathematical paradigm to explain hESC self-organization. The resolution of our three aims will have a strong impact on our understanding of the dynamic integration of signaling pathways underlying human cell fate specification with direct relevance to both basic understanding, and clinical applications of hESCs.

Project Summary/Abstract ! Most genetic recorders described so far utilize the CRISPR/Cas9 system to leave a unique genetic scar in each cell that can be traced in daughter cells. However, since these systems are largely deletion based, there is a finite number of events and lineages that they can be used to trace. Hence all these recorders are fundamentally restricted in the number of lineages they can trace since development in mammals is a prolonged process, with radial glia in the brain dividing greater than 50 times. This central limitation precludes the utility of these approaches in mammalian model organisms that are most relevant to human development. Moreover, these technologies do not allow dynamic recording of biological events throughout the life-cycle of an animal. Thus, a different approach to lineage-tracing and biological recording is needed that does not reach saturation and will permit chronicling lineages in whole organisms and complex tissues. Here we propose to exploit recently described base-editors to enable continuous, dynamic, genetic recording in non-human primates (NHPs), such as a marmoset. Our strategy, called CHRONICLE, bypasses many of the limitations of currently used systems. CHRONICLE (Cellular Hierarchy Recording in Organisms by Nucleotide Interconversion with Cas9 Linked Editors) combines base-editing with self-targeting guide RNA arrays to generate a large repertoire of sequence variants that can be used to trace cellular hierarchy lineages in complex mammalian brains. This tool combined with single cell RNA sequencing (scRNA) will enable us to probe the developmental differences between the human and marmoset cortex and the lineage trajectory of cell types found in each species and the circuits they might contribute to. In Aim 1, we propose to use CHRONICLE to trace lineages in vitro in cortical organoids derived from human embryonic stem cells (hESCs) to trace the intrinsic properties of neural progenitors in a controlled environment. In Aim 2, we will introduce CHRONICLE into marmoset cortical organoids, derived from marmoset induced pluripotent stem cells (iPSCs), as well as marmoset pre-neurulation embryos. This will allow a comparison between in vitro and in vivo development. Finally, in Aim 3, we will use the data obtained from Aims 1 and 2 to compare developmental lineage tree in the marmoset cortex by phylogenetic reconstruction of CHRONICLE evolution in projection neurons, interneurons, and glia using scRNA analysis. This approach will permit chronicling lineages dynamically in a primate brain and bypass many of the limitations of current CRISPR-based molecular recorders. Since many stages of cortical development are unique to primates, this study has the potential to reveal novel lineage relationships and developmental milestones for the first time, and provide answers to key developmental questions. Solving the lineage tree of a primate brain will impact our ability to utilize developmental principles to restore function to diseased and damaged tissues.

Project Summary/Abstract How accurate cell fate specification occurs in the context of dynamic tissue-scale rearrangements is one of the most exciting questions in developmental biology. In this proposal we aim at deciphering the interplay between fate acquisition, patterning and morphogenesis of the ectodermal germ layer in the context of human neurulation. We have recently developed a robust protocol which allows for the generation of extremely reproducible human neuruloids: self-organized human Embryonic Stem Cell (hESC) assemblies that recapitulate the organization of the ectodermal compartment at neurulation stages by organizing neural, neural crest, placodes and epidermis populations within the same colony on adhesive micropatterns. This self-organization is extremely reproducible and can be quantified with sub-cellular resolution and in real time over hundreds of colonies. Armed with this novel technology, we propose three specific aims. The first is to unravel the mechanism of cell-cell signaling driving self-organization. The second is to integrate signaling with fate acquisition and morphogenesis through live reporter imaging and time dependent single cell RNAseq. Finally, the third aim focuses on the full characterization of the origin and sub-populations of ectodermal derivatives and their in vivo validation by performing side by side comparisons with stage-matched marmoset fetal samples and grafting experiments in chick embryos. The generation of large numbers of homogenous human neuruloids, where self-organization of ectodermal fate can be followed dynamically for a period of one week, with sub-cellular resolution, not only solves the inherent heterogeneity observed in cerebral organoids, but provides us a unique opportunity to study these events in models of human embryos. This will have a high impact in both basic research as well as clinical application, a prospect already on the horizon. !

ABSTRACT The common marmoset provides a very relevant primate model for understanding the organization of the human nervous system and the diseases that affect it. Like humans, marmosets also demonstrate cooperative social behavior and have advanced cognitive processes, making them of great interest in the field for modeling developmental and psychiatric diseases and their therapies. They are also ideal for multigenerational genetic experiments as they give birth twice a year and mature faster than most primates. However, while the CRISPR/Cas9 system has been used to knockout genes and create knock-ins of single amino acids in a heritable manner in marmosets, it has been a challenge in the field to create germline transmissible models of gene reporters and trinucleotide repeat genes analogous to their murine counterparts. The very low efficiency of homologous recombination (HR) in primates has precluded knocking-in coding sequences by simply injecting Cas9 protein and a guide RNA into embryos during in vitro fertilization (IVF) as is done for creating knockouts. This limitation has prevented modelling of more genetically complex neurological diseases such as Huntington?s disease (HD) and for creating conditional reporters in marmosets, both of which are mainstays in the mouse neurogenetics field. In addition to low HR frequency, other barriers to creating germline transmission of knock- ins include the absence of a well annotated marmoset genome until recently, lack of protocols for derivation of ground state marmoset pluripotent stem cells (cjPSCs), the low percentage of marmoset pregnancies after embryo reimplantation, and a general deficiency of developmental biology expertise in the marmoset field. We propose to harness our labs? expertise in developmental biology, IVF technologies, and transgenic stem cell biology to overcome this barrier to widespread use of marmosets. We aim to create transgenic knock-in cjPSCs, convert them into ground-state pluripotent stem cells and then inject them into IVF morula to create a chimeric founder marmoset that carries the modified genome. We then aim to screen the transgenic gametes from the founder marmosets to create the F1 progeny and use them to correlate the molecular-behavioral phenotype of HD. As proof-of-principle, we will focus on three knock-in reporter lines to broadly target excitatory, inhibitory, and peripheral neuronal populations. Together, if successful, our aims will result in creation of the first primate model with neuron-specific reporters, establish the marmoset as a valid model of HD, enable access to single- cell transcriptomic changes at the early stages of HD in a primate disease model, and finally correlate these molecular changes with the behavioral phenotype. These aims will provide fundamental insights into the biology of HD and the role of huntingtin protein in different classes of neurons. The outcome of this project will also influence a better understanding of poly-glutamine neurodegenerative diseases that affect humans. In addition, the transgenic marmosets that we generate will be broadly available to the research community and enable new studies into neural circuits, development, behavior, and a wide range of optogenetic applications.